Patent application title: Interleukin-10 Antibodies

Abstract:

The methods and compositions provided herein relate generally to IL-10
specific antibodies and uses thereof. More specifically, compositions of
humanized IL-10 specific antibodies and methods to use such antibodies in
modulating the biological activity of IL-10, particularly in autoimmune
disorders and pathogen-mediated immunopathology.

Claims:

1-9. (canceled)

10. A humanized recombinant antibody molecule that binds IL-10, or binding
fragment thereof, comprising:at least one antibody light chain, or
binding fragment thereof, comprising a polypeptide having at least one
amino acid sequence selected from the group consisting of SEQ ID NO:11 at
CDR1, SEQ ID NO:12 at CDR2, and SEQ ID NO:13 at CDR3; and a framework
region, wherein the amino acid sequence of framework region is all or
substantially all of a human immunoglobin amino acid sequence; andat
least one antibody heavy chain, or binding fragment thereof, comprising a
polypeptide having at least one amino acid sequence selected of SEQ ID
NO:15 at CDR1, SEQ ID NO:16 at CDR2, and SEQ ID NO:17 at CDR3; and a
framework region, wherein the amino acid sequence of framework region is
all or substantially all of a human immunoglobin amino acid sequence.

25. A method of suppressing an immune response in a human subject
comprising administering to a subject in need thereof, an antibody
specific for IL-10, or a binding fragment thereof, in an amount effective
to block the biological activity of IL-10, wherein the antibody is the
antibody of claim 10 or 16.

26. The method of claim 25, wherein the immune response is a humoral
response.

27. The method of claim 25, wherein the subject has systemic lupus
erythematosus.

32. The method of claim 25, further comprising administering an
immunosuppressive agent.

33-34. (canceled)

35. A composition comprising an antibody, or binding fragment thereof, in
combination with a pharmaceutically acceptable carrier or diluent,
wherein the antibody is the antibody of claim 10 or 16.

36. The composition of claim 35, further comprising an immunosuppressive
agent.

37. (canceled)

38. An isolated nucleic acid encoding the polypeptide of claim 10 or 16.

39-41. (canceled)

42. An expression vector comprising the nucleic acid sequence of claim 38
operably linked to control sequences recognized by a host cell
transfected with the vector.

43. A host cell comprising the vector of claim 42.

44. A method of producing a polypeptide, comprising culturing the host
cell of claim 43 under conditions wherein the nucleic acid sequence is
expressed, thereby producing the polypeptide, and recovering the
polypeptide from the host cell.

45-46. (canceled)

47. An isolated nucleic acid sequence encoding an antibody specific for
IL-10 comprising a light chain having the nucleic acid sequence of SEQ ID
NO:21 and a heavy chain having the nucleic acid sequence of SEQ ID NO:22.

48. The nucleic acid of claim 47, wherein light chain has an ATCC deposit
number of PTA-5927 and the heavy chain has an ATCC deposit number of
PTA-5926.

49. (canceled)

50. An isolated nucleic acid sequence encoding a binding fragment of the
antibody encoded by the nucleic acid sequence of claim 47.

51. The nucleic acid of claim 50, wherein the binding fragment is an
antibody fragment selected from the group consisting of Fab, Fab',
Fab'-SH, Fv, scFv, F(ab')2, and a diabody.

52. A method to identify an acceptor germline sequence for a humanized
antibody, which method comprises the steps of:a) identifying a non-human
antibody that has the desired biological activity;b) determining the
amino acid sequence of a non-human antibody VH and VL domains;
andc) comparing the nonhuman antibody sequence to a group of human
germline sequences, wherein the comparison comprises the substeps of:1)
assigning the sequence of non-human VH and VL domain sequences
residue numbers;2) delineating the CDR and FR regions in the sequence;3)
assigning a predetermined numerical score at each residue position for
which the non-human and human germline sequences are identical; and4)
totaling all of the residue scores to generate a total score for each
human germline sequence; andd) identifying the human germline sequence
with the highest total residue score as the acceptor germline sequence.

53. The method of claim 52, further comprising the substeps of:a)
assigning a numerical score of 1 for each residue position for which the
non-human and human germline sequences are identical that was not scored
in substep (3) to germline sequences with identical total residue scores
after substep (4);b) totaling all of the residue scores to generate a
total score for each human germline sequence.

54. The method of claim 52, wherein the VH region is scored as in
Table 2.

55. The method of claim 52, wherein the VL region is scored as in
Table 3.

56. The method of claim 52, wherein the non-human antibody is specific for
IL-10 and inhibits the biological activity of IL-10.

57. An antibody having an acceptor germline sequence identified by the
method of claim 52.

Description:

[0001]This application is a Divisional of U.S. patent application Ser. No.
11/623,006, filed on Jan. 12, 2007, now U.S. Pat. No. 7,662,379, issued
on Feb. 16, 2010, which is a Continuation of U.S. patent application Ser.
No. 10/985,584, filed Nov. 9, 2004, now abandoned, which claims benefit
from U.S. Provisional Patent Application No. 60/518,999, filed Nov. 10,
2003, each of which is incorporated herein by reference in its entirety.

[0004]Multiple pathogens, particularly intracellular pathogens, elicit
IL-10 production to slow or completely stall the effective removal of the
pathogen by the immune response. Moore et al., Annu. Rev. Immunol. 11:165
(1993). For example, in blood lymphocytes from patients with HIV,
leprosy, or tuberculosis, peripheral blood lymphocytes are typically
anergic or nonresponsive in vitro when challenged with the pathogen.
However, the neutralization of IL-10 in these demonstrated that an active
effector response, i.e., Th1 reactivity, was present in these cells.
Thus, it is believed that IL-10 is effectively commandeered by the
pathogen to facilitate its infective state.

[0005]IL-10 is also associated with autoimmunity in vivo. Autoimmunity
results from the development from autoantibodies, autoreactive T cells,
or some combination thereof that target normal tissue. One example of
autoimmune disease is systemic lupus erythematosus (SLE), a chronic
rheumatic disease in which connective tissue throughout the body becomes
inflamed. Autoantibodies that attack normal body tissue as if it were an
outside invade result in the characteristic inflammation. While the
precise cause is not fully understood, researchers believe it has both
genetic and environmental components. Specifically, B-cell hyperactivity
and the presence of various autoantibodies characterize SLE. Typically,
IgG autoantibodies reactive to double stranded DNA (IgG anti-dsDNA abs)
are elevated in patients with SLE. Between 60 and 70% of SLE patients
produce IgG anti-dsDNA abs, some of which are nephrotoxic. SLE is ten
times more prevalent in women than men, with symptoms ranging from facial
rashes to disabling and potentially life-threatening organ dysfunction.
It can develop at any age, but is most common in young adults.

[0006]Numerous studies support a role for IL-10 in the pathology
associated with SLE. For example, while IL-10 is typically not produced
by cells without appropriate stimulation, both B cells and macrophages
from SLE patients spontaneously produce high levels of IL-10 in vitro.
Llorente, et al., Arthritis Rheum. 40:249-60 (1997). In several studies,
researchers demonstrated a correlation between serum levels of IL-10 and
disease activity. Moreover, both in vivo and in vitro studies
demonstrated that the blockade of IL-10 production can alleviate the
clinical manifestations of SLE. See, e.g., Gonzalez-Amaro, et al. J.
Autoimmunity 11:395-402 (1998).

[0007]To date, one of the manifestations of SLE, lupus nephritis, has been
treated with through the use of immunosuppressive therapies, e.g.,
corticosteriods and cyclophosphamides. Although effective, these
therapies are non-specific and substantial toxicities exist which prevent
long term therapy. Thus, specific neutralizing antibodies may be
effective antagonists of IL-10, permitting the removal of the suppressive
effects of IL-10 while leaving the remainder of the immune response
network intact.

[0008]The most significant limitation in using antibodies as a therapeutic
agent in vivo is the immunogenicity of the antibodies. As most monoclonal
antibodies are derived from rodents, repeated use in humans results in
the generation of an immune response against the therapeutic antibody.
Such an immune response results in a loss of therapeutic efficacy at a
minimum and a potential fatal anaphylactic response at a maximum. Initial
efforts to reduce the immunogenicity of rodent antibodies involved the
production of chimeric antibodies, in which mouse variable regions were
fused with human constant regions. Liu et al., Proc. Natl. Acad. Sci. USA
84:3439 (1987). However, mice injected with hybrids of human variable
regions and mouse constant regions develop a strong anti-antibody
response directed against the human variable region, suggesting that the
retention of the entire rodent Fv region in such chimeric antibodies may
still result in unwanted immunogenicity in patients.

[0009]It is generally believed that complementarity determining region
(CDR) loops of variable domains comprise the binding site of antibody
molecules. Therefore, the grafting of rodent CDR loops onto human
frameworks (i.e., humanization) was attempted to further minimize rodent
sequences. Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science
239:1534 (1988). However, CDR loop exchanges still do not uniformly
result in an antibody with the same binding properties as the antibody of
origin. Changes in framework residues (FR), residues involved in CDR loop
support, in humanized antibodies also are required to preserve antigen
binding affinity. Kabat et al., J. Immunol. 147:1709 (1991). While the
use of CDR grafting and framework residue preservation in a number of
humanized antibody constructs has been reported, it is difficult to
predict if a particular sequence will result in the antibody with the
desired binding, and sometimes biological, properties. See, e.g., Queen
et al., Proc. Natl. Acad. Sci. USA 86:10029 (1989), Gorman et al., Proc.
Natl. Acad. Sci. USA 88:4181 (1991), and Hodgson, Bio/Technology 9:421
(1991). Moreover, most prior studies used different human sequences for
animal light and heavy variable sequences, rendering the predictive
nature of such studies questionable. Sequences of known antibodies have
been used or, more typically, those of antibodies having known X-ray
structures, antibodies NEW and KOL. See, e.g., Jones et al., supra;
Verhoeyen et al., supra; and Gorman et al., supra. Exact sequence
information has been reported for only a few humanized constructs.

[0010]The present invention provides humanized monoclonal antibodies which
recognize human IL-10 and modulate its activity, in particular with
regard to autoimmune disorders. The humanized antibody should provide an
alternative therapy choice without the toxicity and non-specificity
associated with current treatments.

BRIEF SUMMARY OF THE INVENTION

[0011]Provided herein is a humanized recombinant antibody molecule that
binds IL-10, or binding fragment thereof, comprising: at least one
antibody light chain variable region, or binding fragment thereof,
comprising a polypeptide having at least one amino acid sequence selected
from the group consisting of SEQ ID NO:1 at CDR1, SEQ ID NO:2 at CDR2,
and SEQ ID NO:3 at CDR3; and a framework region, wherein the amino acid
sequence of framework region is all or substantially all of a human
immunoglobin amino acid sequence; and at least one antibody heavy chain
variable region, or binding fragment thereof, comprising a polypeptide
having at least one amino acid sequence selected from the group of SEQ ID
NO:6 at CDR1, SEQ ID NO:7 at CDR2, and SEQ ID NO:8 at CDR3; and a
framework region, wherein the amino acid sequence of framework region is
all or substantially all of a human immunoglobin amino acid sequence.
Also provided herein is an antibody, wherein the antibody light chain, or
binding fragment thereof, comprises a polypeptide having a variable
region of SEQ ID NO:4. In one specific embodiment, the antibody light
chain, or binding fragment thereof, comprises a polypeptide having a
variable region and a constant region of SEQ ID NO:5. In one specific
embodiment, the antibody heavy chain, or binding fragment thereof,
comprises a polypeptide having a variable region of SEQ ID NO:9. In
another specific embodiment, the antibody heavy chain, or binding
fragment thereof, comprises a polypeptide having a variable region and a
constant region of SEQ ID NO:10.

[0012]Further provided herein is a chimeric recombinant antibody molecule
that binds IL-10 or binding fragment thereof, comprising: at least one
antibody light chain variable region, or binding fragment thereof,
comprising a polypeptide having at least one amino acid sequence selected
from the group consisting of SEQ ID NO:1 at CDR1, SEQ ID NO:2 at CDR2,
and SEQ ID NO:3 at CDR3; and at least one antibody heavy chain variable
region, or binding fragment thereof, comprising a polypeptide having at
least one amino acid sequence selected from the group consisting of SEQ
ID NO:6 at CDR1, SEQ ID NO:7 at CDR2, and SEQ ID NO:8 at CDR3.

[0013]Also provided herein is a humanized recombinant antibody molecule
that binds IL-10, or binding fragment thereof, comprising: at least one
antibody light chain, or binding fragment thereof, comprising a
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:11 at CDR1, SEQ ID NO:12 at CDR2, and SEQ ID
NO:13 at CDR3; and a framework region, wherein the amino acid sequence of
framework region is all or substantially all of a human immunoglobin
amino acid sequence; and at least one antibody heavy chain, or binding
fragment thereof, comprising a polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NO:15 at CDR1, SEQ ID NO:16
at CDR2, and SEQ ID NO:17 at CDR3; and a framework region, wherein the
amino acid sequence of framework region is all or substantially all of a
human immunoglobin amino acid sequence. In one specific embodiment, the
antibody light chain, or binding fragment thereof, comprises a
polypeptide having a variable region and a constant region of SEQ ID
NO:14. In yet another specific embodiment, the antibody heavy chain, or
binding fragment thereof, comprises a polypeptide having a variable
region and a constant region of SEQ ID NO:18.

[0014]Further provided herein is a chimeric recombinant antibody molecule
that binds IL-10, or binding fragment thereof, comprising: at least one
antibody light chain, or binding fragment thereof, comprising a
polypeptide having at least one amino acid sequence selected from the
group consisting of SEQ ID NO:11 at CDR1, SEQ ID NO:12 at CDR2, and SEQ
ID NO:13 at CDR3; and at least one antibody heavy chain, or binding
fragment thereof, comprising a polypeptide having at least one amino acid
sequence selected from the group consisting of SEQ ID NO:15 at CDR1, SEQ
ID NO:16 at CDR2, and SEQ ID NO:17 at CDR3.

[0015]In one embodiment, the antibodies described supra further comprise a
heavy chain constant region, wherein the heavy chain constant region
comprises a γ1, γ2, γ3, or γ4 human heavy chain
constant region or a variant thereof. In one embodiment, the antibodies
described above further comprise a light chain constant region, wherein
the light chain constant region comprises a lambda or a kappa human light
chain constant region. In some embodiments, the binding fragment of these
antibodies is an antibody fragment selected from the group consisting of
Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, and a diabody.

[0016]Further provided herein is a method of suppressing an immune
response in a human subject comprising administering to a subject in need
thereof an antibody specific for IL-10, or a binding fragment thereof, in
an amount effective to block the biological activity of IL-10, wherein
the antibody is an antibody disclosed herein. The immune response
suppressed by this method is a humoral or a cellular response. In one
embodiment, the subject treated by this method has systemic lupus
erythematosus. In another embodiment, the subject has immune
thrombocytopenic purpura (ITC). In yet another embodiment, the subject
has lupus nephritis. In a further embodiment, the subject has HIV. In
another embodiment, the subject has hepatitis C. In one specific
embodiment, the method of suppressing an immune response in a human
subject comprising administering to a subject in need thereof (1) an
antibody specific for IL-10, or a binding fragment thereof, in an amount
effective to block the biological activity of IL-10, wherein the antibody
is an antibody disclosed herein, and (2) an immunosuppressive agent.

[0017]Provided herein is a composition comprising an antibody, or binding
fragment thereof, in combination with a pharmaceutically acceptable
carrier or diluent, wherein the antibody is one of the antibodies
disclosed supra.

[0018]Further provided herein is an isolated nucleic acid encoding the
polypeptide of the antibodies disclosed supra. Also provided herein is an
expression vector comprising the isolated nucleic acid sequence operably
linked to control sequences recognized by a host cell transfected with
the vector. Provided herein is a host cell comprising the vector
comprising the isolated nucleic acid sequence. Further provided herein is
a method of producing a polypeptide, comprising culturing the host cell
comprising the vector under conditions wherein the nucleic acid sequence
is expressed, thereby producing the polypeptide, and recovering the
polypeptide from the host cell.

[0019]Provided herein is an isolated nucleic acid sequence encoding an
antibody specific for IL-10 comprising a light chain having the nucleic
acid sequence of SEQ ID NO:19 and a heavy chain having the nucleic acid
sequence of SEQ ID NO:20. In further embodiments, the light chain has an
American Type Culture Collection (ATCC) deposit number of PTA-5923 and
the heavy chain has an ATCC deposit number of PTA-5922.

[0020]Provided herein is an isolated nucleic acid sequence encoding an
antibody specific for IL-10 comprising a light chain having the nucleic
acid sequence of SEQ ID NO:21 and a heavy chain having the nucleic acid
sequence of SEQ ID NO:22. In a further embodiment, the light chain has an
ATCC deposit number of PTA-5927 and the heavy chain has an ATCC deposit
number of PTA-5926.

[0021]Further provided herein is an isolated nucleic acid sequence
encoding a binding fragment of the antibody encoded by the above nucleic
acid sequences. In one embodiment, the binding fragment is an antibody
fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv,
scFv, and F(ab')2.

[0022]Provided herein is a method to identify an acceptor germline
sequence for a humanized antibody, which method comprises the steps of:
a) identifying a non-human antibody that has the desired biological
activity; b) determining the amino acid sequence of a non-human antibody
VH and VL domains; and c) comparing the nonhuman antibody
sequence to a group of human germline sequences, wherein the comparison
comprises the substeps of: 1) assigning the sequence of non-human VH
and VL domain sequences residue numbers; 2) delineating the CDR and
FR regions in the sequence; 3) assigning a predetermined numerical score
at each residue position for which the non-human and human germline
sequences are identical; and 4) totaling all of the residue scores to
generate a total score for each human germline sequence; and d)
identifying the human germline sequence with the highest total residue
score as the acceptor germline sequence. In one embodiment, the method
further comprises the substeps of: 5) assigning a numerical score of 1
for each residue position for which the non-human and human germline
sequences are identical that was not scored in substep (3) to germline
sequences with identical total residue scores after substep (4); 6)
totaling all of the residue scores to generate a total score for each
human germline sequence. In a specific embodiment, the non-human antibody
is specific for IL-10 and inhibits the biological activity of IL-10. In a
specific embodiment, the numerical scores are assigned to the residues as
in Tables 2 and 3 for VH and VL regions, respectively.

[0023]Further provided herein is an antibody generated by the above
method.

[0028]FIG. 2A is a concentration-time profile for 12G8 antibody
administered i.v. as described in Example III.

[0029]FIG. 2B is a concentration-time profile for 12G8 administered s.c.
as described in Example III.

[0030]FIG. 3A shows that administration of the humanized anti-IL-10
antibody, SCH708980, confers resistance to Leishmania major infection in
IL-10 transgenic mice. Infection was determined by measuring footpad
swelling with a caliper at the times indicated. 12G8 antibody was
administered as described in Example VI.

[0031]FIG. 3B shows that administration of the rat anti-IL-10 antibody,
12G8, confers resistance to Leishmania major infection in IL-10
transgenic mice. Infection was determined by measuring footpad swelling
with a caliper at the times indicated. 12G8 antibody was administered as
described in Example VI.

DETAILED DESCRIPTION OF THE INVENTION

[0032]For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the subsections
that follow.

A. DEFINITIONS

[0033]Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as is commonly understood by one of ordinary
skill in the art to which this invention belongs. All patents,
applications, published applications and other publications referred to
herein are incorporated by reference in their entirety. If a definition
set forth in this section is contrary to or otherwise inconsistent with a
definition set forth in the patents, applications, published applications
and other publications that are herein incorporated by reference, the
definition set forth in this section prevails over the definition that is
incorporated herein by reference.

[0035]As used herein, the term "antibody" refers to any form of antibody
or fragment thereof that exhibits the desired biological activity. Thus,
it is used in the broadest sense and specifically covers monoclonal
antibodies (including full length monoclonal antibodies), polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies), and
antibody fragments so long as they exhibit the desired biological
activity.

[0036]As used herein, the term "IL-10 binding fragment" or "binding
fragment thereof" encompasses a fragment or a derivative of an antibody
that still substantially retain its biological activity of inhibiting
IL-10 activity. Therefore, the term "antibody fragment" or IL-10 binding
fragment refers to a portion of a full length antibody, generally the
antigen binding or variable region thereof. Examples of antibody
fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies;
linear antibodies; single-chain antibody molecules, e.g., sc-Fv; and
multispecific antibodies formed from antibody fragments. Typically, a
binding fragment or derivative retains at least 50% of its IL-10
inhibitory activity. Preferably, a binding fragment or derivative retains
at least 60%, 70%, 80%, 90%, 95%, 99% or 100% of its IL-10 inhibitory
activity. It is also intended that a IL-10 binding fragment can include
conservative amino acid substitutions that do not substantially alter its
biologic activity.

[0037]The term "monoclonal antibody", as used herein, refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population are
identical except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly specific,
being directed against a single antigenic epitope. In contrast,
conventional (polyclonal) antibody preparations typically include a
multitude of antibodies directed against (or specific for) different
epitopes. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of the
antibody by any particular method. For example, the monoclonal antibodies
to be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature 256: 495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat.
No. 4,816,567). The "monoclonal antibodies" may also be isolated from
phage antibody libraries using the techniques described in Clackson et
al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222:
581-597 (1991), for example.

[0038]The monoclonal antibodies herein specifically include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a particular
antibody class or subclass, while the remainder of the chain(s) is
identical with or homologous to corresponding sequences in antibodies
derived from another species or belonging to another antibody class or
subclass, as well as fragments of such antibodies, so long as they
exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and
Morrison et al., Proc. Natl. Acad Sci. USA 81: 6851-6855 (1984)).

[0039]As used herein, the term "single-chain Fv" or "scFv" antibody refers
to antibody fragments comprising the VH and VL domains of
antibody, wherein these domains are present in a single polypeptide
chain. Generally, the Fv polypeptide further comprises a polypeptide
linker between the VH and VL domains which enables the sFv to
form the desired structure for antigen binding. For a review of sFv, see
Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg
and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0040]As used herein, the term "diabodies" refers to small antibody
fragments with two antigen-binding sites, which fragments comprise a
heavy chain variable domain (VH) connected to a light chain variable
domain (VL) in the same polypeptide chain (VH-VL). By
using a linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites. Diabodies
are described more fully in, e.g., EP 404,097; WO 93/11161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0041]As used herein, the term "humanized antibody" refers to forms of
antibodies that contain sequences from non-human (e.g., murine)
antibodies as well as human antibodies. Such antibodies are chimeric
antibodies which contain minimal sequence derived from non-human
immunoglobulin. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable domains,
in which all or substantially all of the hypervariable loops correspond
to those of a non-human immunoglobulin and all or substantially all of
the FR regions are those of a human immunoglobulin sequence. The
humanized antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin.

[0042]As used herein, the term "hypervariable region" refers to the amino
acid residues of an antibody which are responsible for antigen-binding.
The hypervariable region comprises amino acid residues from a
"complementarity determining region" or "CDR" (i.e. residues 24-34 (L1),
50-56 (L2) and 89-97 (L3) in the light chain variable domain and residues
31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable
domain; Kabat et al., Sequences of Proteins of Immunological Interest,
5th Ed. Public Health Service, National Institutes of Health, Bethesda,
Md. (1991)) and/or those residues from a "hypervariable loop" (i.e.
residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain
variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy
chain variable domain; Chothia and Lesk, J. Mol. Biol. 196: 901-917
(1987)). As used herein, the term "framework" or "FR" residues refers to
those variable domain residues other than the hypervariable region
residues defined herein as CDR residues.

[0043]As used herein, the term "conservative substitution" refers to
substitutions of amino acids are known to those of skill in this art and
may be made generally without altering the biological activity of the
resulting molecule. Those of skill in this art recognize that, in
general, single amino acid substitutions in non-essential regions of a
polypeptide do not substantially alter biological activity (see, e.g.,
Watson, et al., Molecular Biology of the Gene, The Benjamin/Cummings Pub.
Co., p. 224 (4th Edition 1987)). Such exemplary substitutions are
preferably made in accordance with those set forth in TABLE 1 as follows:

[0044]Other substitutions are also permissible and may be determined
empirically or in accord with known conservative substitutions.

[0045]As used herein, the term "isolated nucleic acid molecule" refers to
a nucleic acid molecule that is identified and separated from at least
one contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the antibody nucleic acid. An
isolated nucleic acid molecule is other than in the form or setting in
which it is found in nature. Isolated nucleic acid molecules therefore
are distinguished from the nucleic acid molecule as it exists in natural
cells. However, an isolated nucleic acid molecule includes a nucleic acid
molecule contained in cells that ordinarily express the antibody where,
for example, the nucleic acid molecule is in a chromosomal location
different from that of natural cells.

[0046]The expression "control sequences" refers to DNA sequences necessary
for the expression of an operably linked coding sequence in a particular
host organism. The control sequences that are suitable for prokaryotes,
for example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and enhancers.

[0047]Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For example,
DNA for a presequence or secretory leader is operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or enhancer is operably linked
to a coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if it is
positioned so as to facilitate translation. Generally, "operably linked"
means that the DNA sequences being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers are used in accordance with
conventional practice.

[0048]As used herein, the expressions "cell," "cell line," and "cell
culture" are used interchangeably and all such designations include
progeny. Thus, the words "transformants" and "transformed cells" include
the primary subject cell and cultures derived therefrom without regard
for the number of transfers. It is also understood that all progeny may
not be precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function or
biological activity as screened for in the originally transformed cell
are included. Where distinct designations are intended, it will be clear
from the context.

[0049]As used herein, "polymerase chain reaction" or "PCR" refers to a
procedure or technique in which minute amounts of a specific piece of
nucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S.
Pat. No. 4,683,195. Generally, sequence information from the ends of the
region of interest or beyond needs to be available, such that
oligonucleotide primers can be designed; these primers will be identical
or similar in sequence to opposite strands of the template to be
amplified. The 5' terminal nucleotides of the two primers can coincide
with the ends of the amplified material. PCR can be used to amplify
specific RNA sequences, specific DNA sequences from total genomic DNA,
and cDNA transcribed from total cellular RNA, bacteriophage or plasmid
sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp.
Quant. Biol. 51:263 (1987); Erlich, ed., PCR TECHNOLOGY (Stockton Press,
N.Y., 1989). As used herein, PCR is considered to be one, but not the
only, example of a nucleic acid polymerase reaction method for amplifying
a nucleic acid test sample comprising the use of a known nucleic acid as
a primer and a nucleic acid polymerase to amplify or generate a specific
piece of nucleic acid.

[0050]As used herein, the term "germline sequence" refers to a sequence of
unrearranged immunoglobulin DNA sequences. Any suitable source of
unrearranged immunoglobulin may be used.

[0051]As used herein, the term "immunosuppressive agent" refers to natural
or synthetic agents that suppress or modulate an immune response. The
immune response can be a humoral or cellular response.

B. IL-10 SPECIFIC ANTIBODIES

[0052]The compositions and methods disclosed herein relate to the
modulation of IL-10 activity, particularly in immune responses.
Specifically, the compositions and methods herein employ antibodies
specific for the cytokine, IL-10. IL-10 is a potent cytokine that
modulates T and B cell responses through the regulation of growth,
differentiation, and cytokine synthesis of a variety of cell types
involved in immune responses. Notably, IL-10 production is frequently
associated with autoimmune diseases and pathogen-induced immunopathology.
Therefore, a composition, and methods thereof, that modulates and
inhibits IL-10 activity can alter the development and sustainment of
autoimmune disease and related symptoms and ameliorate or reduce
pathogen-associated immunopathology.

[0053]Targeting IL-10 activity with humanized antibodies offers several
unique advantages. First, targeting IL-10 with antibody permits a
specific suppression of IL-10 activity while leaving the remainder of the
immune response intact. In many cases of pathogen-induced
immunopathology, the reduction or elimination of IL-10 activity should
permit the desired effector immune response to eliminate with pathogen
without further pathology. For the autoimmune patient, the reduction or
elimination of IL-10 activity should reduce or eliminate the disease
and/or its symptoms while maintaining the patient's immune competence.
Second, humanized IL-10 antibodies circumvents the limitation associated
with immunogenic rodent antibodies. The use of human sequences eliminates
the immunogenicity of the exogenously administered antibodies, allowing
therapeutic administration.

[0054]Humanized antibodies contain sequences from non-human as well as
human antibodies. Typically, the process of humanization begins with the
generation of a non-human antibody that has the desired biological
activity, i.e., inhibits IL-10 activity. Once a non-human antibody with
the appropriate characteristics is identified, recombinant means are then
employed to create a hybrid sequence using non-human and human sequences.

C. GENERATION OF IL-10 SPECIFIC ANTIBODIES

[0055]Any suitable method for generating monoclonal antibodies may be
used. For example, a recipient may be immunized with IL-10 or a fragment
thereof. Any suitable method of immunization can be used. Such methods
can include adjuvants, other immunostimulants, repeated booster
immunizations, and the use of one or more immunization routes.

[0056]Any suitable source of IL-10 can be used as the immunogen for the
generation of the non-human antibody of the compositions and methods
disclosed herein. Such forms include, but are not limited whole protein,
peptide(s), and epitopes, generated through recombinant, synthetic,
chemical or enzymatic degradation means known in the art. IL-10 is an
acid-sensitive, noncovalent homodimer of two interpenetrating polypeptide
chain. The cytokine is 160 amino acids in length with well conserved
sequences that include an α-helical bundle structure similar to
interferons and hemopoietic cytokines Human and murine IL-10 have 73%
amino acid homology, with human IL-10 being active on murine and human
cells. IL-10 is commercially available or can be produced using well
known molecular biology techniques. Genbank cDNA sequences are available
for the human, pig-tailed macaque, mangabey, rhesus, and owl monkeys,
lemur, mouse, rat, guinea pig, Syrian hamster, rabbit, cat, dog, as well
as others. Recombinant human IL-10 is a 17-18 kDa polypeptide that is not
N-glycosylated.

[0057]Any form of the antigen can be used to generate the antibody that is
sufficient to generate a biologically active antibody. Thus, the
eliciting antigen may be a single epitope, multiple epitopes, or the
entire protein alone or in combination with one or more immunogenicity
enhancing agents known in the art. The eliciting antigen may be an
isolated full-length protein, a cell surface protein (e.g., immunizing
with cells transfected with at least a portion of the antigen), or a
soluble protein (e.g., immunizing with only the extracellular domain
portion of the protein). The antigen may be produced in a genetically
modified cell. The DNA encoding the antigen may genomic or non-genomic
(e.g., cDNA) and encodes at least a portion of the extracellular domain.
As used herein, the term "portion" refers to the minimal number of amino
acids or nucleic acids, as appropriate, to constitute an immunogenic
epitope of the antigen of interest. Any genetic vectors suitable for
transformation of the cells of interest may be employed, including but
not limited to adenoviral vectors, plasmids, and non-viral vectors, such
as cationic lipids.

[0058]Any suitable method can be used to elicit an antibody with the
desired biologic properties to inhibit IL-10. It is desirable to prepare
monoclonal antibodies (mAbs) from various mammalian hosts, such as mice,
rodents, primates, humans, etc. Description of techniques for preparing
such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)
BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications, Los
Altos, Calif., and references cited therein; Harlow and Lane (1988)
ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL
ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York,
N.Y. Thus, monoclonal antibodies may be obtained by a variety of
techniques familiar to researchers skilled in the art. Typically, spleen
cells from an animal immunized with a desired antigen are immortalized,
commonly by fusion with a myeloma cell. See Kohler and Milstein (1976)
Eur. J. Immunol. 6:511-519. Alternative methods of immortalization
include transformation with Epstein Barr Virus, oncogenes, or
retroviruses, or other methods known in the art. See, e.g., Doyle, et al.
(eds. 1994 and periodic supplements) CELL AND TISSUE CULTURE: LABORATORY
PROCEDURES, John Wiley and Sons, New York, N.Y. Colonies arising from
single immortalized cells are screened for production of antibodies of
the desired specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells may be enhanced by various
techniques, including injection into the peritoneal cavity of a
vertebrate host. Alternatively, one may isolate DNA sequences which
encode a monoclonal antibody or a binding fragment thereof by screening a
DNA library from human B cells according, e.g., to the general protocol
outlined by Huse, et al. (1989) Science 246:1275-1281.

[0059]Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, e.g., Huse et al., Science
246:1275-1281 (1989); and Ward et al., Nature 341:544-546 (1989). The
polypeptides and antibodies of the present invention may be used with or
without modification, including chimeric or humanized antibodies.
Frequently, the polypeptides and antibodies will be labeled by joining,
either covalently or non-covalently, a substance which provides for a
detectable signal. A wide variety of labels and conjugation techniques
are known and are reported extensively in both the scientific and patent
literature. Suitable labels include radionuclides, enzymes, substrates,
cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins
may be produced, see Cabilly U.S. Pat. No. 4,816,567; and Queen et al.
(1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; or made in transgenic
mice, see Mendez et al. (1997) Nature Genetics 15:146-156; also see
Abgenix and Medarex technologies.

[0060]Antibodies or binding compositions against predetermined fragments
of IL-10 can be raised by immunization of animals with conjugates of the
polypeptide, fragments, peptides, or epitopes with carrier proteins.
Monoclonal antibodies are prepared from cells secreting the desired
antibody. These antibodies can be screened for binding to normal or
defective IL-10. These monoclonal antibodies will usually bind with at
least a Kd of about 1 μM, more usually at least about 300 nM,
typically at least about 30 nM, preferably at least about 10 nM, more
preferably at least about 3 nM or better, usually determined by ELISA.
Suitable non-human antibodies may also be identified using the biologic
assays described in Section D infra.

C. HUMANIZATION OF IL-10 SPECIFIC ANTIBODIES

[0061]Any suitable non-human antibody can be used as a source for the
hypervariable region. Sources for non-human antibodies include, but are
not limited to, murine, lupine, bovine, and primates. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody) in
which hypervariable region residues of the recipient are replaced by
hypervariable region residues from a non-human species (donor antibody)
such as mouse, rat, rabbit or nonhuman primate having the desired
specificity, affinity, and capacity. In some instances, Fv framework
region (FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized antibodies may
comprise residues which are not found in the recipient antibody or in the
donor antibody. These modifications are made to further refine antibody
performance of the desired biological activity. For further details, see
Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332:
323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0063]Amino acid sequence variants of humanized anti-IL-10 antibody are
prepared by introducing appropriate nucleotide changes into the humanized
anti-IL-10 antibody DNA, or by peptide synthesis. Such variants include,
for example, deletions from, and/or insertions into and/or substitutions
of, residues within the amino acid sequences shown for the humanized
anti-IL-10 F(ab) (e.g. as in SEQ ID NO's 5 and 10). Any combination of
deletion, insertion, and substitution is made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid changes also may alter post-translational
processes of the humanized anti-IL-10 antibody, such as changing the
number or position of glycosylation sites.

[0064]A useful method for identification of certain residues or regions of
the humanized anti-IL-10 antibody polypeptide that are preferred
locations for mutagenesis is called "alanine scanning mutagenesis," as
described by Cunningham and Wells, Science 244: 1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a neutral
or negatively charged amino acid (most preferably alanine or polyalanine)
to affect the interaction of the amino acids with IL-10 antigen. The
amino acid residues demonstrating functional sensitivity to the
substitutions then are refined by introducing further or other variants
at, or for, the sites of substitution. Thus, while the site for
introducing an amino acid sequence variation is predetermined, the nature
of the mutation per se need not be predetermined. For example, to analyze
the performance of a mutation at a given site, ala scanning or random
mutagenesis is conducted at the target codon or region and the expressed
humanized anti-IL-10 antibody variants are screened for the desired
activity.

[0065]Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include humanized anti-IL-10 antibody
with an N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the humanized anti-IL-10 antibody
molecule include the fusion to the N- or C-terminus of humanized
anti-IL-10 antibody of an enzyme or a polypeptide which increases the
serum half-life of the antibody.

[0066]Another type of variant is an amino acid substitution variant. These
variants have at least one amino acid residue in the humanized anti-IL-10
antibody molecule removed and a different residue inserted in its place.
The sites of greatest interest for substitutional mutagenesis include the
hypervariable loops, but FR alterations are also contemplated. Tables 2
and 3 in the method described below provides guidance as to hypervariable
region residues which can be altered. Hypervariable region residues or FR
residues involved in antigen binding are generally substituted in a
relatively conservative manner.

[0067]Another type of amino acid variant of the antibody alters the
original glycosylation pattern of the antibody. By altering is meant
deleting one or more carbohydrate moieties found in the antibody, and/or
adding one or more glycosylation sites that are not present in the
antibody. Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate moiety to
the side chain of an asparagine residue. The tripeptide sequences
asparagine-X-serine and asparagine-X-threonine, where X is any amino acid
except proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the presence
of either of these tripeptide sequences in a polypeptide creates a
potential glycosylation site. O-linked glycosylation refers to the
attachment of one of the sugars N-aceylgalactosamine, galactose, or
xylose to a hydroxyamino acid, most commonly serine or threonine,
although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0068]Addition of glycosylation sites to the antibody is conveniently
accomplished by altering the amino acid sequence such that it contains
one or more of the above-described tripeptide sequences (for N-linked
glycosylation sites). The alteration may also be made by the addition of,
or substitution by, one or more serine or threonine residues to the
sequence of the original antibody (for O-linked glycosylation sites).

[0069]Nucleic acid molecules encoding amino acid sequence variants of
humanized IL-10 specific antibody are prepared by a variety of methods
known in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring amino
acid sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of
an earlier prepared variant or a non-variant version of humanized
anti-IL-10 antibody.

[0070]Ordinarily, amino acid sequence variants of the humanized anti-IL-10
antibody will have an amino acid sequence having at least 75% amino acid
sequence identity with the original humanized antibody amino acid
sequences of either the heavy or the light chain (e.g. as in SEQ ID NO:5
and 10), more preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, and most preferably at least 95%. Identity or
homology with respect to this sequence is defined herein as the
percentage of amino acid residues in the candidate sequence that are
identical with the humanized anti-IL-10 residues, after aligning the
sequences and introducing gaps, if necessary, to achieve the maximum
percent sequence identity, and not considering any conservative
substitutions as part of the sequence identity. None of N-terminal,
C-terminal, or internal extensions, deletions, or insertions into the
antibody sequence shall be construed as affecting sequence identity or
homology.

[0071]The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, the
antibody is a IgG antibody. Any isotype of IgG can be used, including
IgG1, IgG2, IgG3, and IgG4. Variants of the IgG
isotypes are also contemplated. The humanized antibody may comprise
sequences from more than one class or isotype. Optimization of the
necessary constant domain sequences to generate the desired biologic
activity is readily achieved by screening the antibodies in the
biological assays described below.

[0072]Likewise, either class of light chain can be used in the
compositions and methods herein. Specifically, kappa, lambda, or variants
thereof are useful in the present compositions and methods.

[0073]Any suitable portion of the CDR sequences from the non-human
antibody can be used. The CDR sequences can be mutagenized by
substitution, insertion or deletion of at least one residue such that the
CDR sequence is distinct from the human and non-human antibody sequence
employed. It is contemplated that such mutations would be minimal.
Typically, at least 75% of the humanized antibody residues will
correspond to those of the non-human CDR residues, more often 90%, and
most preferably greater than 95%.

[0074]Any suitable portion of the FR sequences from the human antibody can
be used. The FR sequences can be mutagenized by substitution, insertion
or deletion of at least one residue such that the FR sequence is distinct
from the human and non-human antibody sequence employed. It is
contemplated that such mutations would be minimal. Typically, at least
75% of the humanized antibody residues will correspond to those of the
human FR residues, more often 90%, and most preferably greater than 95%.

[0075]CDR and FR residues are determined according to the standard
sequence definition of Kabat. Kabat et al., Sequences of Proteins of
Immunological Interest, National Institutes of Health, Bethesda Md.
(1987).

[0076]Provided herein is a method to identify an acceptor germline
sequence for a humanized antibody, which method comprises the steps of:
a) identifying a non-human antibody that has the desired biological
activity; b) determining the amino acid sequence of a non-human antibody
VH and VL domains; and c) comparing the nonhuman antibody
sequence to a group of human germline sequences, wherein the comparison
comprises the substeps of: 1) assigning the non-human V sequences residue
numbers according to Kabat supra; 2) delineating the CDR and FR regions
in the sequence according to Kabat supra; 3) assigning a predetermined
numerical score at specific residue position for which the non-human and
human antibody germline sequences are identical; and 4) totaling all of
the residue scores to generate a total score for each human germline
sequence; and d) identifying the human germline sequence with the highest
total residue score as the acceptor germline sequence. In one embodiment,
the method further comprises the substeps of: 5) assigning a numerical
score of 1 for each FR residue position for which the non-human and human
antibody germline sequences are identical that was not scored in substep
(3) to germline sequences with identical total residue scores after
substep (4); 6) totaling all of the residue scores to generate a total
score for each human germline sequence. In a specific embodiment, the
non-human antibody is specific for IL-10 and inhibits the biological
activity of IL-10. Also provided herein is an antibody generated by the
above method.

[0077]In one embodiment, the IL-10 antibody is humanized using the
following method. First, the non-human VL and VH domains of the
IL-10 antibody are cloned and sequenced, and the amino acid sequence
determined. Then, the non-human VH sequence are compared to a group
of five human VH germline amino acid sequences. The five groups
contain one representative from the subgroups IGHV1 and IGHV4 and three
representatives from subgroup IGHV3. The VH subgroups are listed in
M.-P. Lefranc, Exp. Clin. Immunogenetics, 18:100-116 (2001).
Specifically, the comparison with the five germline sequences begins with
the assignment of residue numbers to the non-human VH sequence
according to the Kabat numbering system. See Kabat, et al., U.S.
Department of Health and Human Services, NIH Pub. 91-3242 (5th Ed.,
1991). The non-human VH sequence are then aligned with each of the
five human germline sequences. Since the V genes only comprise VH
residues 1-94, only these residues are considered in the alignment. Next,
the complementarity-determining (CDR) and framework (FR) regions in the
sequence are delineated. CDR and FR are delineated according to the
combination of the definitions provided in Kabat, et al., U.S. Department
of Health and Human Services, NIH Pub. 91-3242 (5th Ed., 1991), and C.
Chothia & A.M. Lesk, J. Mol. Biol., 196:901-917 (1987). Therefore, the
CDR definition used is residues 26-35 for CDR1, residues 50-65 for CDR2,
and CDR3 is residues 95-102 for CDR3 of the VH domain. The next step
involves assigning a numerical score at identified residue position where
the non-human and human sequences are identical. One example of this
scoring is shown in Table 2 below.

[0078]After the residue positions are assigned a numerical score, all of
the residue scores are totaled. The acceptor germline sequence is the one
with the highest total score. In a case where two or more germline
sequences have identical scores, then add 1 to the total for each
position where the non-human and human sequences are IDENTICAL for the
following residues: 1, 3, 5-23, 25, 36, 38, 40-43, 46, 66, 68, 70, 72,
74, 75, 77, 79-90, and 92 (max 49). The residue scores are totaled again,
and the acceptor germline sequence is the one with the highest total
score. If two or more germline sequences still have identical scores,
either one can be used as the acceptor germline sequence.

[0079]If the VL sequence is a member of the kappa subclass of
VL, the non-human VL sequence from the IL-10 specific antibody
is compared to a group of four human VL kappa germline amino acid
sequences. The four sequences are comprised of one representative from
each of four established human VL subgroups listed in V. Barbie &
M.-P. Lefranc, Exp. Clin. Immunogenetics 15:171-183 (1998) and M.-P.
Lefranc, Exp. Clin. Immunogenetics 18:161-174 (2001). The four sequences
also correspond to the four subgroups listed in Kabat et al., U.S.
Department of Health and Human Services, NIH Pub. 91-3242, pp. 103-130
(5th Ed., 1991). The comparison of the non-human sequence to the four
germline sequences begins with the assignment of residue numbers to the
non-human VL sequence residues according to Kabat et al., U.S.
Department of Health and Human Services, NIH Pub. 91-3242 (5th Ed.,
1991). The non-human VL sequences are then aligned with each of the
four human germline sequences. Since the V genes only comprise VL
residues 1-95, only these residues are considered in the alignment. Next,
the complementarity-determining (CDR) and framework (FR) regions are
delineated in the sequence. CDR and FR are delineated according to the
combination of the definitions provided in Kabat et al., U.S. Department
of Health and Human Services, NIH Pub. 91-3242 (5th Ed. 1991), and C.
Chothia & A. M. Lesk, J. Mol. Biol., 196:901-917 (1987). Therefore, the
CDR definition used is residues 24-34 for CDR1, residues 50-56 for CDR2,
and residues 89-97 for CDR3 of the VL domain. The next step involves
assigning a numerical score at identified residue position where the
non-human and human sequences are identical. One example of this scoring
is shown in Table 3 below.

[0080]After the residue positions are assigned a numerical score, all of
the residue scores are totaled. The acceptor germline sequence is the one
with the highest total score. In a case where two or more germline
sequences have identical scores, then add 1 to the total for each
position where the non-human and human sequences are IDENTICAL for the
following residues: 1, 3, 5-23, 35, 37, 39-42, 57, 59-61, 63, 65-70,
72-86, and 88. The residue scores are totaled again, and the acceptor
germline sequence is the one with the highest total score. If two or more
germline sequences still have identical scores, either one can be used as
the acceptor germline sequence.

[0081]For recombinant production of the antibody, the nucleic acid
encoding it is isolated and inserted into a replicable vector for further
cloning (amplification of the DNA) or for expression. DNA encoding the
monoclonal antibody is readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable of
binding specifically to genes encoding the heavy and light chains of the
antibody). Many vectors are available. The vector components generally
include, but are not limited to, one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an enhancer
element, a promoter, and a transcription termination sequence.

[0082]In one embodiment, the antibody is a humanized recombinant antibody
molecule that binds IL-10, or binding fragment thereof, comprising: at
least one antibody light chain variable region, or binding fragment
thereof, comprising a polypeptide having at least one amino acid sequence
selected from the group consisting of SEQ ID NO:1 (KTSQNIFENLA) at CDR1,
SEQ ID NO:2 (NASPLQA) at CDR2, and SEQ ID NO:3 (HQYYSGYT) at CDR3; and a
framework region, wherein the amino acid sequence of framework region is
all or substantially all of a human immunoglobin amino acid sequence; and
at least one antibody heavy chain variable region, or binding fragment
thereof, comprising a polypeptide having at least one amino acid sequence
selected from the group consisting of SEQ ID NO:6 (GFTFSDYHMA) at CDR1,
SEQ ID NO:7 (SITLDATYTYYRDSVRG) at CDR2, SEQ ID NO:8 (HRGFSVWLDY) at
CDR3; and a framework region, wherein the amino acid sequence of
framework region is all or substantially all of a human immunoglobin
amino acid sequence. In a specific embodiment, the antibody light chain,
or binding fragment thereof, comprises a polypeptide having a variable
region of SEQ ID NO:4 (DIQMTQSPSSLSASVGDRVTITCKTSQNIFENLAWYQQKPGKAPKLLIY
NASPLQAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYSG-YTFGPGTKLELKRT). In one
specific embodiment, the antibody light chain, or binding fragment
thereof, comprises a polypeptide having a variable region and a constant
region of SEQ ID NO:5. See Table 4. In one specific embodiment, the
antibody heavy chain, or binding fragment thereof, comprises a
polypeptide having a variable region of SEQ ID NO:9
(QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYHMAWV
RQAPGKGLEWVASITLDATYTYYRDSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYY
CARHRGFSVWLDYWGQGTLVTVSS). In another specific embodiment, the antibody
heavy chain, or binding fragment thereof, comprises a polypeptide having
a variable region and a constant region of SEQ ID NO:10. See Table 5.

[0083]Plasmids containing the nucleic acids encoding the humanized 12G8
light and heavy chains were deposited with the American Type Culture
Collection (ATCC) as deposit numbers PTA-5923 and PTA-5922, respectively.

[0084]In one embodiment, the antibody is a humanized recombinant antibody
molecule that binds IL-10, or binding fragment thereof, comprising: at
least one antibody light chain, or binding fragment thereof, comprising a
polypeptide having at least one amino acid sequence selected from the
group consisting of SEQ ID NO:11 (RASESVDDYGHSFMH) at CDR1, SEQ ID NO:12
(RASTLES) at CDR2, and SEQ ID NO:13 (QQGNEDPWT) at CDR3; and a framework
region, wherein the amino acid sequence of framework region is all or
substantially all of a human immunoglobin amino acid sequence; and at
least one antibody heavy chain, or binding fragment thereof, comprising a
polypeptide having at least one amino acid sequence selected from the
group consisting of SEQ ID NO:15 (GFSLTNYGVH) at CDR1, SEQ ID NO:16
(VIWSGGSTDYNAAFIS) at CDR2, and SEQ ID NO:17 (NRGYDVYFDY) at CDR3; and a
framework region, wherein the amino acid sequence of framework region is
all or substantially all of a human immunoglobin amino acid sequence. In
one specific embodiment, the antibody light chain, or binding fragment
thereof, comprises a polypeptide having a variable region and a constant
region of SEQ ID NO:14. See Table 6. In yet another specific embodiment,
the antibody heavy chain, or binding fragment thereof, comprises a
polypeptide having a variable region and a constant region of SEQ ID
NO:18. See Table 7.

[0085]Plasmids containing the humanized 11D8 heavy and light chains were
deposited with the ATCC as deposit numbers PTA-5926 and PTA-5927,
respectively.

[0086]In one embodiment, the antibodies described supra further comprise a
heavy chain constant region, wherein the heavy chain constant region
comprises a γ1, γ2, γ3, or γ4 human heavy chain
constant region or a variant thereof. In one embodiment, the antibodies
described above further comprise a light chain constant region, wherein
the light chain constant region comprises a lambda or a kappa human light
chain constant region. In some embodiments, the binding fragment of these
antibodies is an antibody fragment selected from the group consisting of
Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, and a diabody.

[0087]Also provided herein is a chimeric recombinant antibody molecule
that binds IL-10 or binding fragment thereof, comprising: at least one
antibody light chain variable region, or binding fragment thereof,
comprising a polypeptide having at least one amino acid sequence selected
from the group consisting of SEQ ID NO:1 at CDR1, SEQ ID NO:2 at CDR2,
and SEQ ID NO:3 at CDR3; and at least one antibody heavy chain variable
region, or binding fragment thereof, comprising a polypeptide having at
least one amino acid sequence selected from the group consisting of SEQ
ID NO:6 at CDR1, SEQ ID NO:7 at CDR2, and SEQ ID NO:8 at CDR3.

[0088]In a specific embodiment, the chimeric recombinant antibody molecule
has a light chain as set forth in SEQ ID NO: 23 and a heavy chain as set
forth in SEQ ID NO:24. See Table 8. Nucleic acids encoding the 12G8
chimeric antibody light and heavy chains were deposited at the ATCC as
deposit numbers PTA-5925 and PTA-5924, respectively.

[0089]Further provided herein is a chimeric recombinant antibody molecule
that binds IL-10, or binding fragment thereof, comprising: at least one
antibody light chain, or binding fragment thereof, comprising a
polypeptide having at least one amino acid sequence selected from the
group consisting of SEQ ID NO:11 at CDR1, SEQ ID NO:12 at CDR2, and SEQ
ID NO:13 at CDR3; and at least one antibody heavy chain, or binding
fragment thereof, comprising a polypeptide having at least one amino acid
sequence selected from the group consisting of SEQ ID NO:15 at CDR1, SEQ
ID NO:16 at CDR2, and SEQ ID NO:17 at CDR3.

[0090]Further provided herein is an isolated nucleic acid encoding the
polypeptide of the antibodies disclosed supra. Also provided herein is an
expression vector comprising the isolated nucleic acid sequence operably
linked to control sequences recognized by a host cell transfected with
the vector. Provided herein is a host cell comprising the vector
comprising the isolated nucleic acid sequence. Further provided herein is
a method of producing a polypeptide, comprising culturing the host cell
comprising the vector under conditions wherein the nucleic acid sequence
is expressed, thereby producing the polypeptide, and recovering the
polypeptide from the host cell.

[0091]Provided herein is an isolated nucleic acid sequence encoding an
antibody specific for IL-10 comprising a light chain having the nucleic
acid sequence of SEQ ID NO:19 and a heavy chain having the nucleic acid
sequence of SEQ ID NO:20. See Tables 4 and 5.

[0092]Provided herein is an isolated nucleic acid sequence encoding an
antibody specific for IL-10 comprising a light chain having the nucleic
acid sequence of SEQ ID NO:21 and a heavy chain having the nucleic acid
sequence of SEQ ID NO:22. See Tables 6 and 7.

[0093]Further provided herein is an isolated nucleic acid sequence
encoding a binding fragment of the antibody encoded by the above nucleic
acid sequences. In one embodiment, the binding fragment is an antibody
fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv,
scFv, F(ab')2, and a diabody.

[0094]Bispecific antibodies are also useful in the present methods and
compositions. As used herein, the term "bispecific antibody" refers to an
antibody, typically a monoclonal antibody, having binding specificities
for at least two different antigenic epitopes. In one embodiment, the
epitopes are from the same antigen. In another embodiment, the epitopes
are from two different antigens. Methods for making bispecific antibodies
are known in the art. For example, bispecific antibodies can be produced
recombinantly using the co-expression of two immunoglobulin heavy
chain/light chain pairs. See, e.g., Milstein et al., Nature 305: 537-39
(1983). Alternatively, bispecific antibodies can be prepared using
chemical linkage. See, e.g., Brennan, et al., Science 229: 81 (1985).
Bispecific antibodies include bispecific antibody fragments. See, e.g.,
Hollinger, et al., Proc. Natl. Acad. Sci. U.S.A. 90: 6444-48 (1993),
Gruber, et al., J. Immunol. 152: 5368 (1994).

D. BIOLOGICAL ACTIVITY OF HUMANIZED ANTI-IL-10 ANTIBODIES

[0095]Antibodies having the characteristics identified herein as being
desirable in a humanized anti-IL-10 antibody can be screened for
inhibitory biologic activity in vitro or suitable binding affinity. To
screen for antibodies which bind to the epitope on human IL-10 bound by
an antibody of interest (e.g., those which block binding of the cytokine
to its receptor), a routine cross-blocking assay such as that described
in ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Ed
Harlow and David Lane (1988), can be performed. Alternatively, epitope
mapping, e.g., as described in Champe et al., J. Biol. Chem. 270:
1388-1394 (1995), can be performed to determine whether the antibody
binds an epitope of interest. Antibody affinities (e.g. for human IL-10)
may be determined using standard Scatchard analysis. Preferred humanized
antibodies are those which bind human IL-10 with a Kd value of no
more than about 1×10-7; preferably no more than about
1×10-8; more preferably no more than about 1×10-9;
and most preferably no more than about 2×10-10.

[0096]The antibodies and fragments thereof useful in the present
compositions and methods are biologically active antibodies and
fragments. As used herein, the term "biologically active" refers to an
antibody or antibody fragment that is capable of binding the desired the
antigenic epitope and directly or indirectly exerting a biologic effect.
Typically, these effects result from the failure of IL-10 to bind its
receptor. As used herein, the term "specific" refers to the selective
binding of the antibody to the target antigen epitope. Antibodies can be
tested for specificity of binding by comparing binding to IL-10 to
binding to irrelevant antigen or antigen mixture under a given set of
conditions. If the antibody binds to IL-10 at least 10, and preferably 50
times more than to irrelevant antigen or antigen mixture then it is
considered to be specific.

[0097]The inhibitory IL-10 specific antibody can inhibit its biological
activity in any manner, including but not limited to production of IL-1,
IFN-γ, PGE2, IL-12, TNF, CC and CXC chemokines, and the cell
surface expression of MHC class II antigens, CD54, CD80, and CD86. The
biologic activity of an IL-10 specific antibody can be determined by any
useful method. See, e.g., U.S. Pat. Nos. 6,239,260 and 6,207,154. In one
example, the biologic activity is assessed in cell proliferation assay
using the murine mast cell line, MC9/2. See Thompson-Snipes et al., J.
Exp. Med. 173:507-10 (1991). IL-10 stimulates the proliferation of this
cell line, and therefore an inhibitory antibody can be identified by its
ability to reduce proliferation. The ED50 for proliferation of the
MC9/2 cell line is typically 0.5-1.0 ng/mL. An antibody is inhibitory for
proliferation if it inhibits the proliferation of cells relative to the
proliferation of cells in the absence of the antibody or in the presence
of a non-binding antibody. Proliferation may be quantified using any
suitable methods. Typically, the proliferation is determined by assessing
the incorporation of radioactive-labeled nucleotides into DNA (e.g.,
3H-thymidine). In another embodiment, proliferation is determined by
ATP luminescence. Preferably, the antibody useful in the present
compositions inhibits 80% of IL-10's biologic activity, more preferably
95%, most preferably 99%.

E. USES OF HUMANIZED ANTI-IL-10 ANTIBODIES

[0098]Provided herein is a method of suppressing an immune response in a
human subject comprising administering to a subject in need thereof an
antibody specific for IL-10, or a binding fragment thereof, in an amount
effective to block the biological activity of IL-10, wherein the antibody
is a humanized recombinant antibody molecule that binds IL-10, or binding
fragment thereof, comprising: at least one antibody light chain variable
region, or binding fragment thereof, comprising a polypeptide having at
least one amino acid sequence selected from the group consisting of SEQ
ID NO:1 at CDR1, SEQ ID NO:2 at CDR2, and SEQ ID NO:3 at CDR3; and a
framework region, wherein the amino acid sequence of framework region is
all or substantially all of a human immunoglobin amino acid sequence; and
at least one antibody heavy chain variable region, or binding fragment
thereof, comprising a polypeptide having at least one amino acid sequence
selected from the group consisting of SEQ ID NO:6 at CDR1, SEQ ID NO:7 at
CDR2, and SEQ ID NO:8 at CDR3; and a framework region, wherein the amino
acid sequence of framework region is all or substantially all of a human
immunoglobin amino acid sequence.

[0099]Further provided herein is a method of suppressing an immune
response in a human subject comprising administering to a subject in need
thereof an antibody specific for IL-10, or a binding fragment thereof, in
an amount effective to block the biological activity of IL-10, wherein
the antibody is a humanized recombinant antibody molecule that binds
IL-10, or binding fragment thereof, comprising: at least one antibody
light chain, or binding fragment thereof, comprising a polypeptide having
at least one amino acid sequence selected from the group consisting of
SEQ ID NO:11 at CDR1, SEQ ID NO:12 at CDR2, and SEQ ID NO:13 at CDR3; and
a framework region, wherein the amino acid sequence of framework region
is all or substantially all of a human immunoglobin amino acid sequence;
and at least one antibody heavy chain, or binding fragment thereof,
comprising a polypeptide having at least one amino acid sequence selected
from the group consisting of SEQ ID NO:15 at CDR1, SEQ ID NO:16 at CDR2,
and SEQ ID NO:17 at CDR3; and a framework region, wherein the amino acid
sequence of framework region is all or substantially all of a human
immunoglobin amino acid sequence.

[0100]The immune response suppressed by these methods is a humoral or a
cellular response. The suppression of the humoral and cellular responses
can be determined using well known methods in the art. For example, in
diseases associated with high levels of autoreactive antibodies, e.g.,
SLE, a reduction in the serum levels of these antibodies relative to the
pre-treatment serum levels is an indication of the suppression of the
humoral response. Likewise, the suppression of the cellular immune
response can be determined using well known in vitro analyses, e.g.,
proliferation and cytotoxicity assays or characterization of activation
phenotypes of peripheral blood lymphocytes by, e.g., flow cytometric
analysis. See CURRENT PROTOCOLS IN IMMUNOLOGY, most recent edition. In
one embodiment, the subject treated by this method has systemic lupus
erythematosus. In another embodiment, the subject has immune
thrombocytopenic purpura (ITC). In yet another embodiment, the subject
has lupus nephritis. In a further embodiment, the subject has HIV. In
another embodiment, the subject has hepatitis C.

[0101]Provided herein is a composition comprising an antibody, or binding
fragment thereof, in combination with a pharmaceutically acceptable
carrier or diluent, wherein the antibody is one of the antibodies
disclosed herein.

[0102]Any subject that would benefit from treatment with IL-10 specific
antibodies can be treated using the compositions and methods provided
herein. Any subject can be treated with the methods and compositions
provided herein. Such a subject is a mammal, preferably a human, with an
autoimmune disease or symptom or pathogen-induced immunopathology. In one
specific embodiment, the subject has SLE, lupus nephritis, rheumatoid
arthritis, ITC, HIV or hepatitis C. Veterinary uses of the disclosed
methods and compositions are also contemplated.

[0103]As used herein, "inhibit" or "treat" or "treatment" includes a
postponement of development of the symptoms associated with autoimmune
disease or pathogen-induced immunopathology and/or a reduction in the
severity of such symptoms that will or are expected to develop. The terms
further include ameliorating existing uncontrolled or unwanted
autoimmune-related or pathogen-induced immunopathology symptoms,
preventing additional symptoms, and ameliorating or preventing the
underlying causes of such symptoms. Thus, the terms denote that a
beneficial result has been conferred on a vertebrate subject with an
autoimmune or pathogen-induced immunopathology disease or symptom, or
with the potential to develop such a disease or symptom.

[0104]As used herein, the term "therapeutically effective amount" or
"effective amount" refers to an amount of an IL-10 specific antibody that
when administered alone or in combination with an additional therapeutic
agent to a cell, tissue, or subject is effective to prevent or ameliorate
the autoimmune disease or pathogen-induced immunopathology associated
disease or condition or the progression of the disease. A therapeutically
effective dose further refers to that amount of the compound sufficient
to result in amelioration of symptoms, e.g., treatment, healing,
prevention or amelioration of the relevant medical condition, or an
increase in rate of treatment, healing, prevention or amelioration of
such conditions. When applied to an individual active ingredient
administered alone, a therapeutically effective dose refers to that
ingredient alone. When applied to a combination, a therapeutically
effective dose refers to combined amounts of the active ingredients that
result in the therapeutic effect, whether administered in combination,
serially or simultaneously.

[0105]An antibody useful in the present methods (from whatever source
derived, including without limitation from recombinant and
non-recombinant sources) may be administered to a subject in need, by
itself, or in pharmaceutical compositions where it is mixed with suitable
carriers or excipient(s) at doses to treat or ameliorate a variety of
disorders. Such a composition may also contain (in addition to protein
and a carrier) diluents, fillers, salts, buffers, stabilizers,
solubilizers, and other materials well known in the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the effectiveness of the biological activity of the active
ingredient(s). The characteristics of the carrier will depend on the
route of administration.

[0106]The pharmaceutical composition of the invention may also contain
other immunosuppressive or immunomodulating agents. Any suitable
immunosuppressive agent can be employed, including but not limited to
anti-inflammatory agents, corticosteroids, cyclosporine, tacrolimus
(i.e., FK-506), sirolimus, interferons, soluble cytokine receptors (e.g.,
sTNRF and sIL-1R), agents that neutralize cytokine activity (e.g.,
inflixmab, etanercept), mycophenolate mofetil, 15-deoxyspergualin,
thalidomide, glatiramer, azathioprine, leflunomide, cyclophosphamide,
methotrexate, and the like. The pharmaceutical composition can also be
employed with other therapeutic modalities such as phototherapy and
radiation.

[0107]In another embodiment, kits are provided that contain the necessary
reagents to carry out the assays of the methods provided herein.
Specifically provided herein is a compartment kit comprising one or more
containers, wherein a first container comprises one or more antibodies
specific to IL-10, and one or more other containers comprising one or
more of the following: reconstitution reagents, administration reagents.
The containers can be glass, plastic, or strips of plastic or paper. In
one embodiment, the kit also contain written instructions.

[0108]In practicing the methods of treatment or use provided herein, a
therapeutically effective amount of antibody provided herein is
administered to a mammal having a condition suitable for treatment with
IL-10. The antibody may be administered in accordance with the methods
herein either alone or in combination with other therapies such as
treatments employing other immunomodulating factors (e.g., cytokines),
immunosuppressive agents, and the like. When co-administered with one or
more biologically active agents, the antibody provided herein may be
administered either simultaneously with the biologically active agent(s),
or sequentially. If administered sequentially, the attending physician
will decide on the appropriate sequence of administering protein of the
present invention in combination with the biologically active agent(s).

[0109]Toxicity and therapeutic efficacy of the antibody compositions,
administered alone or in combination with an immunosuppressive agent, can
be determined by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and it can
be expressed as the ratio between LD50 and ED50. Antibodies
exhibiting high therapeutic indices are preferred. The data obtained from
these cell culture assays and animal studies can be used in formulating a
range of dosage for use in human. The dosage of such compounds lies
preferably within a range of circulating concentrations that include the
ED50 with little or no toxicity. The dosage may vary within this
range depending upon the dosage form employed and the route of
administration utilized.

[0110]Techniques for formulation and administration of the antibodies of
the instant methods may be found in REMINGTON'S PHARMACEUTICAL SCIENCES,
Mack Publishing Co., Easton, Pa., latest edition. The mode of
administration is not particularly important. Suitable routes of
administration may, for example, include oral, rectal, transmucosal, or
intestinal administration; parenteral delivery, including intramuscular,
subcutaneous, intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections. Administration of antibody used in the
pharmaceutical composition or to practice the method of the present
invention can be carried out in a variety of conventional ways, such as
oral ingestion, inhalation, topical application or cutaneous,
subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous
injection. Intravenous administration to the patient is preferred.

[0111]Alternately, one may administer the antibody in a local rather than
systemic manner, for example, via injection of the antibody directly into
an arthritic joint or pathogen-induced lesion characterized by
immunopathology, often in a depot or sustained release formulation.
Furthermore, one may administer the antibody in a targeted drug delivery
system, for example, in a liposome coated with a tissue-specific
antibody, targeting, for example, arthritic joint or pathogen-induced
lesion characterized by immunopathology. The liposomes will be targeted
to and taken up selectively by the afflicted tissue.

[0112]Pharmaceutical compositions for use in accordance with the present
methods thus may be formulated in a conventional manner using one or more
physiologically acceptable carriers comprising excipients and auxiliaries
that facilitate processing of the active compounds into preparations that
can be used pharmaceutically. These pharmaceutical compositions may be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping or lyophilizing processes. Proper
formulation is dependent upon the route of administration chosen. When
administered in liquid form, a liquid carrier such as water, petroleum,
oils of animal or plant origin such as peanut oil, mineral oil, soybean
oil, or sesame oil, or synthetic oils may be added. The liquid form of
the pharmaceutical composition may further contain physiological saline
solution, dextrose or other saccharide solution, or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When
administered in liquid form, the pharmaceutical composition contains from
about 0.5 to 90% by weight of protein of the present invention, and
preferably from about 1 to 50% protein of the present invention.

[0113]When a therapeutically effective amount of antibody of the methods
herein is administered by intravenous, cutaneous or subcutaneous
injection, protein of the present invention will be in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The preparation
of such parenterally acceptable protein solutions, having due regard to
pH, isotonicity, stability, and the like, is within the skill in the art.
A preferred pharmaceutical composition for intravenous, cutaneous, or
subcutaneous injection should contain, in addition to protein of the
present invention, an isotonic vehicle such as Sodium Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the
art. The pharmaceutical composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of skill in the art. For injection, the agents
of the invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution, Ringer's
solution, or physiological saline buffer. For transmucosal
administration, penetrants appropriate to the barrier to be permeated are
used in the formulation. Such penetrants are generally known in the art.

[0114]For administration by inhalation, the antibodies for use according
to the present methods are conveniently delivered in the form of an
aerosol spray presentation from pressurized packs or a nebuliser, with
the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
other suitable gas. In the case of a pressurized aerosol the dosage unit
may be determined by providing a valve to deliver a metered amount.
Capsules and cartridges of, e.g., gelatin for use in an inhaler or
insufflator may be formulated containing a powder mix of the compound and
a suitable powder base such as lactose or starch. The compounds may be
formulated for parenteral administration by injection, e.g., by bolus
injection or continuous infusion. Formulations for injection may be
presented in unit dosage form, e.g., in ampules or in multi-dose
containers, with an added preservative. The compositions may take such
forms as suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing and/or
dispersing agents.

[0115]Pharmaceutical formulations for parenteral administration include
aqueous solutions of the active compounds in water-soluble form.
Additionally, suspensions of the active compounds may be prepared as
appropriate oily injection suspensions. Suitable lipophilic solvents or
vehicles include fatty oils such as sesame oil, or synthetic fatty acid
esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous
injection suspensions may contain substances that increase the viscosity
of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or
dextran. Optionally, the suspension may also contain suitable stabilizers
or agents that increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions. Alternatively, the active
ingredient may be in powder form for constitution with a suitable
vehicle, e.g., sterile pyrogen-free water, before use.

[0116]The amount of antibody useful in the disclosed methods in the
pharmaceutical composition of the present invention will depend upon the
nature and severity of the condition being treated, and on the nature of
prior treatments that the patient has undergone. Ultimately, the
attending physician will decide the amount of protein of the present
invention with which to treat each individual patient. It is to be
expected that the dosage will vary according to the age, weight and
response of the individual patient. Initially, the attending physician
will administer low doses of antibodies of the present methods and
observe the patient's response. Larger doses of antibodies of the present
invention may be administered until the optimal therapeutic effect is
obtained for the patient, and at that point the dosage is not increased
further. It is contemplated that the various pharmaceutical compositions
used to practice the methods herein should contain about 0.01 μg to
about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably
about 0.1 μg to about 1 mg) of antibody of the present invention per
kg body weight. When administered, the therapeutic composition for use in
this invention is, of course, in a pyrogen-free, physiologically
acceptable form. Therapeutically useful agents other than an antibody of
the present methods that may also optionally be included in the
composition as described above, may alternatively or additionally, be
administered simultaneously or sequentially with the composition in the
methods of the invention. The exact formulation, route of administration
and dosage can be chosen by the individual physician in view of the
patient's condition. See, e.g., Fingl et al., THE PHARMACOLOGICAL BASIS
OF THERAPEUTICS (latest edition). Dosage amount and interval may be
adjusted individually to provide plasma levels of the active moiety
sufficient to maintain the desired therapeutic effects, or minimal
effective concentration (MEC). The MEC will vary for each compound but
can be estimated from in vitro data; for example, the concentration
necessary to achieve 50-90% inhibition of biologic activity using the
assays described herein.

[0117]The antibody provided herein can be administered alone or in
combination with other therapeutic modalities. The antibody provided
herein also can be administered alone or in combination with other
antibodies identified as inhibitors of IL-10 activity or other
immunosuppressive agents.

[0122]Computer sequence analysis is performed, e.g., using available
software programs, including those from the GCG (U. Wisconsin) and
GenBank sources. Public sequence databases were also used, e.g., from
GenBank and others.

Example II

Humanization of Anti-Human IL-10 Antibodies

[0123]The humanization of the rat anti-human IL-10 antibody, 12G8, was
performed as described in Section C supra. FIG. 1 shows the assignment of
the assignment of residue numbers and corresponding numerical scores for
residue positions that are identical to the germline sequences being
examined. Calculations are shown for the 12G8 variable regions of the
light (FIG. 1A) and heavy (FIG. 1B) of the 12G8 anti-human IL-10 antibody
and for the variable regions of the light (FIG. 1c) and heavy (FIG. 1D)
of the 11D8 anti-human IL-10 antibody.

Example III

Pharmacokinetics of 12G8, an Anti-Human IL-10 Antibody

[0124]Objective: To obtain estimates of in-vivo terminal half-lives and
subcutaneous bioavailability for the 12F8 antibody in a murine model.

[0127]Experimental Design: Mice received a single bolus injection of
antibody either intravenously (i.v. in lateral tail vein) or
subcutaneously (s.c. at nape of neck or mid-scapular or lateral flank).
Antibody doses included 0.03, 0.3, 3.0, and 30 mg/kg per mouse. The mice
were observed for up to 28 days post-injection. During this time period,
mice were weighed and serum samples taken. Serum samples for the 12G8
(SCH 708980) groups (Groups 1-8) were taken at 0.5, 1, 3, 6, 10, 16 hrs,
Day 1, 2, 3, 5, 7, 10, 14, 21, and 28 post-injection using 5 mice/time
point. In the vehicle group (Group 9), serum samples were taken at
pre-injection, 1 hr post-injection, 14 day or 21 day only using 5
mice/time point. Serum IL-10 levels and serum 12G8 antibody levels were
determined using specific ELISAs.

[0132]Concentration-time profiles are shown for 12F8 antibody using
various dosages and routes in FIG. 2.

[0133]Conclusions: The doses were within 20% of nominal for all groups
except lowest dose level. Lower than expected concentrations, probably
due to presence of anti-SCH708980 (humanized 12G8) antibodies were
observed from Day 10 post-injection in groups 7 or 8. (A) IV Bolus
Pharmacokinetics. Half-lives, clearance and distribution volumes are
typical of those seen for other IgG1 monoclonal antibodies. Distribution
volume is approximately equal to or slightly larger than serum volume
suggesting minimal extravascular distribution. The terminal half-lives
ranged from 10 to 17 days. The increase in AUC was generally
dose-proportional suggesting linear PK over the dose range tested. (B) SC
Bolus Pharmacokinetics. The maximum concentrations were generally
dose-proportional and were reached by 1-2 days postdose suggesting
consistent rates and extents of absorption over the dose range tested.
The increase in AUC was generally dose-proportional suggesting linear PK.
The terminal elimination half-lives ranged from 8-14 days, similar to
other IgG1 monoclonal antibodies. The absolute bioavailability was high,
range=70-100%, although the estimates >90% may be high due to
underestimation of IV AUC.

[0134]The equilibrium dissociation constant (Kd) for humanized antibody
SCH 708980 was determined using KinExA 3000 (Sapidyne Instruments Inc.).
KinExA uses the principle of the Kinetic Exclusion Assay method based on
measuring the concentration of uncomplexed antibody in a mixture of
antibody, antigen and antibody-antigen complex. The concentration of free
antibody was measured by exposing the mixture to a solid-phase
immobilized antigen for a very brief period of time. In practice, this
was accomplished by flowing the solution phase antigen-antibody mixture
past antigen coated particles trapped in a flow cell. Data generated by
the instrument were analyzed using custom software. Equilibrium constants
were calculated using a mathematical theory based on the following
assumptions: [0135]1. The binding follows the reversible binding
equation for equilibrium:

[0139]PMMA particles were coated with biotinylated rhIL5 according to
manufacturer's protocols. For biotinylation of rhIL5 EZ-link TFP
PEO-biotin was used according to manufacturer's recommendations (Pierce
bulletin 0874). All experimental procedures were done according to the
KinExA 3000 manual.

[0140]Experimental conditions: All runs were done in duplicate. For hIL-10
runs the following conditions were used:

[0141]Sample volume: 1.5 ml

[0142]Sample flow rate: 0.25 ml/min

[0143]Label volume: 0.5 ml

[0144]Label flow rate: 0.25 ml/min

[0145]mAb conc.: 0.1 nM

[0146]Highest Ag (hIL-10) conc.: 4.0 nM

[0147]Lowest Ag (hIL-10) conc.: 3.91 pM

[0148]Two-fold serial dilutions of the antigen were prepared and mixed
with the antibody at constant concentration. The mixture was incubated
for 2 hours at room temperature (RT) to equilibrate.

[0149]For mIL-10 runs the following conditions were used:

[0150]Sample volume: 0.5 ml

[0151]Sample flow rate: 0.25 ml/min

[0152]Label volume: 0.5 ml

[0153]Label flow rate: 0.25 ml/min

[0154]mAb conc.: 1 nM

[0155]Highest Ag (mIL-10) conc.: 50 nM

[0156]Lowest Ag (mIL-10) conc.: 48.8 pM

[0157]Two-fold serial dilutions of the antigen were prepared and mixed
with the antibody at constant concentration. The mixture was incubated
for 2 hours at RT. to equilibrate.

[0158]For cyno IL-10 runs the following conditions were used:

[0159]Sample volume: 2 ml

[0160]Sample flow rate: 0.25 ml/min

[0161]Label volume: 1 ml

[0162]Label flow rate: 0.25 ml/min

[0163]mAb conc.: 0.1 nM

[0164]Highest Ag (mIL-10) conc.: 5.0 nM

[0165]Lowest Ag (mIL-10) conc.: 4.88 pM

[0166]Two-fold serial dilutions of the antigen were prepared and mixed
with the antibody at constant concentration. The mixture was incubated
for 2 hours at RT to equilibrate.

Application of Competitive Electrochemiluminescence Assay (ECLA) to
Measure Binding of Anti-hIL-10 Monoclonal Antibodies and hIL-10-Ra to
Recombinant IL-10 of Different Origin

[0168]Summary of technology. Electrochemiluminescence technology was
developed by IGEN, Inc (Gaithersburg, Md.) and is employed in the
M-series M8/384 analyzer used in this work. Electrochemiluminescence
technology utilizes a stable ruthenium metal chelate (Ori-TAG) which, in
the presence of tripropylamine (TPA), generates electrochemiluminescence
upon voltage application. Paramagnetic beads, microns in diameter, act as
the solid phase and facilitate rapid assay kinetics. The bead/complex is
channeled through a flow cell and captured at an electrode by magnetic
application. Voltage is applied and resulting electrochemiluminescence is
measured.

[0170]Protocol. 1/3 serial dilutions in 50 microliters of the assay buffer
were made in 96-well microtiter plate for all unlabeled IL-10
preparations (mIL-10, cyno IL-10, hIL-10 dimer, hIL-10 mono) starting
with 3 μg/ml in the first well. All samples were run in duplicates. 50
μl of hIL-10-biotin at 25 ng/ml was added to each well, followed by
the addition of either hIL-10Ra (50 μl at 100 ng/ml) or anti hIL-10
mAb (50 μl at 10 ng/ml). 50 microliters of Ori-Tag conjugated
secondary antibodies was added to each well at 500 ng/ml conc. For
hIL-10Ra, r12G8 and h12G8 the following Ori-Tag conjugated were used
accordingly: anti-FLAG M2-OriTag, anti-rat IgG-OriTag and anti hIgG
1A2-OriTag. Finally to each well 50 μl of Streptavidin-Dynabeads at
0.1 mg/ml was added. After a one hour incubation at room temperature the
plate was processed by the M-series M8/384 analyzer. Percent inhibition
of the signal by unlabeled IL-10 preparations was calculated relative to
the control samples. To plot the data and calculate IC50 the
GraphPad Prism Software was used.

[0173]In vivo neutralizing efficacy of SCH 708980 and JES.12G8 was
evaluated in the Leishmania major model in mice. In this model, CB6F1
mice normally resistant to parasite infection were rendered susceptible
by heterozygosity for a human IL-10 transgene under the control of the
MHC class II promoter. CB6F1 or CB6F1-huIL-10Tg mice were injected s.c.
with SCH 708980 or JES.12G8 weekly beginning three days before s.c.
footpad challenge with 15×106 stationary phase L. major
parasites. Disease progression was monitored by weekly measurements of
footpad swelling. FIG. 3 shows that both SCH708980 (the humanized 12G8)
and the parental rat 12G8 neutralized the protective effect of IL-10 in a
dose-dependent manner.

[0174]Many modifications and variations of this invention can be made
without departing from its spirit and scope, as will be apparent to those
skilled in the art. The specific embodiments described herein are offered
by way of example only, and the invention is to be limited by the terms
of the appended claims, along with the full scope of equivalents to which
such claims are entitled; and the invention is not to be limited by the
specific embodiments that have been presented herein by way of example.

[0175]Citation of the above publications or documents is not intended as
an admission that any of the foregoing is pertinent prior art, nor does
it constitute any admission as to the contents or date of these
publications or documents. U.S. patents and other publications referenced
herein are hereby incorporated by reference.